Effect of epithelial debridement on human cornea proteoglycans
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1 Brazilian Human corneal Journal explant of Medical proteoglycans and Biological Research (2001) 34: ISSN X Short Communication 325 Effect of epithelial debridement on human cornea proteoglycans E.S. Soriano 1, M.S.Q. Campos 2, J.A.K. Aguiar 1 and Y.M. Michelacci 1 Departamentos de 1 Bioquímica and 2 Oftalmologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil Correspondence Y.M. Michelacci Disciplina de Biologia Molecular Departamento de Bioquímica EPM, UNIFESP Rua Três de Maio, São Paulo, SP Brasil Fax: yara.bioq@epm.br Presented at the 6th Brazilian Symposium on Extracellular Matrix - SIMEC, Angra dos Reis, RJ, Brazil, September 24-27, Research supported by CNPq, CAPES and FAPESP. Received October 19, 2000 Accepted February 5, 2001 Abstract Key words Corneal explants De-epithelization Glycosaminoglycan Proteoglycan
2 326 E.S. Soriano et al.
3 Human corneal explant proteoglycans 327
4 328 E.S. Soriano et al. B PG Immunoblot ì í î Toluidine blue Radioautogram Anti-KS Anti-decorin A PG PG M PG PG M M PG PG M PG PG GAG PG PG + M papain GAG GAG M GAG GAG M M GAG GAG M GAG GAG Figure 1 - Agarose gel electrophoresis of proteoglycans and glycosaminoglycans extracted from human corneas. Aliquots (5 µl) of papain-digested proteoglycans (panel A), or intact proteoglycans (PG) and glycosaminoglycans (GAG) (panel B) extracted from human corneas were submitted to agarose gel electrophoresis in 0.05 M 1,3-diaminopropane acetate buffer, ph 9.0, at 5 V/cm for 1 h. The proteoglycans and glycosaminoglycans were fixed in the gel by 0.1% cetyltrimethylammonium bromide and stained with toluidine blue, as previously described (15). The 35 S-labeled compounds were observed by radioautography. Decorin (a dermatan sulfate proteoglycan) and keratan sulfate were also identified by immunoblotting stained with an anti-decorin polyclonal antibody and MST1 (an anti-keratan sulfate monoclonal antibody). M: Mixture of standard glycosaminoglycans containing chondroitin sulfate (), dermatan sulfate (), keratan sulfate (KS), and heparan sulfate ().
5 Human corneal explant proteoglycans 329 A GAG (mg/g tissue wet weight) B S-GAG (cpm/g tissue wet weight)12000 Control Partial de-epithelization (mg/g tissue wet weight) (mg/g tissue wet weight) Total de-epithelization (mg/g tissue wet weight) KS Control (cpm/g tissue wet weight) Partial de-epithelization (cpm/g tissue wet weight) Total de-epithelization (cpm/g tissue wet weight) KS Figure 2 - Total glycosaminoglycans (A) and 35 S-labeled glycosaminoglycans (B) extracted from explants of intact or epithelium-debrided human corneas. Total glycosaminoglycans were stained with toluidine blue and quantified by densitometry. The 35 S-glycosaminoglycans were visualized by radioautography and scraped off the gels, and radioactivity was measured with a liquid scintillation spectrophotometer. Data are reported as means ± SD of 14 experiments. GAG, Total glycosaminoglycan;, dermatan sulfate; KS, keratan sulfate;, heparan sulfate.
6 330 E.S. Soriano et al. References 1. Hay ED (1980). Development of the vertebrate cornea. International Review of Cytology, 63: Heinegard D, Franzen A, Hedbom E & Sommarin Y (1986). Common structures of the core proteins of interstitial proteoglycans. CIBA Foundation Symposium, 124: Blochberger TC, Vergnes J-P, Hempel J & Hassell JR (1992). cdna to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine. Journal of Biological Chemistry, 267: Funderburgh JL, Funderburgh ML, Brown SJ, Vergnes J-P, Hassell JR, Mann MM & Conrad GW (1993). Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique. Journal of Biological Chemistry, 268: Chakravarti S, Stallings RL, SundarRaj N, Cornuet PK & Hassell JR (1995). Primary structure of human lumican (keratan sulfate proteoglycan) and localization of the gene (LUM) to chromosome 12q21.3-q22. Genomics, 27: Corpus LM, Funderburgh JL, Funderburgh ML, Bottomley GS, Prakash S & Conrad GW (1996). Molecular cloning and tissue distribution of keratocan. Bovine corneal keratan sulfate proteoglycan 37A. Journal of Biological Chemistry, 271: Li W, Vergnes J-P, Cornuet PK & Hassell JR (1992). cdna clone to chick corneal chondroitin/dermatan sulfate proteoglycans reveals identity to decorin. Archives of Biochemistry and Biophysics, 296: Chakravarti S, Magnuson T, Lass JH, Jepsen KJ, LaMantia C & Carroll H (1998). Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican. Journal of Cell Biol-
7 Human corneal explant proteoglycans 331 ogy, 141: Cornuet PK, Blochberger TC & Hassell JR (1994). Molecular polymorphism of lumican during corneal development. Investigative Ophthalmology and Visual Science, 35: Hahn RA & Birk DE (1992). ß-D-xyloside alters dermatan sulfate synthesis and the organization of the developing avian corneal stroma. Development, 115: Nakazawa K, Suzuki S, Wada K & Nakazawa K (1995). Proteoglycan synthesis by corneal explants from developing embryonic chicken. Journal of Biochemistry, 117: Funderburgh JL, Stenzel-Johnson PR & Chandler JW (1983). Corneal glycosaminoglycan synthesis in long-term organ culture. Investigative Ophthalmology and Visual Science, 24: Nakazawa K, Isomura T & Shimoeda S (1995). Proteoglycan synthesis by chicken corneal explants. Journal of Biochemistry, 117: Tai GH, Nisduszynski IA, Fullwood NJ & Huckerby TN (1997). Human corneal keratan sulfates. Journal of Biological Chemistry, 272: Soriano ES, Queiroz MC & Michelacci YM (2000). Glycosaminoglycan synthesis by human corneal explants. Effect of epithelial debridement. Clinica Chimica Acta, 295: Cássaro CMF & Dietrich CP (1977). Distribution of sulfated mucopolysaccharides in invertebrates. Journal of Biological Chemistry, 252: Alves MLM, Straus AH, Takahashi HK & Michelacci YM (1994). Production and characterization of monoclonal antibodies to shark cartilage proteoglycan. Brazilian Journal of Medical and Biological Research, 27: Balduini C, Brovelli A & Castellani AA (1970). Biosynthesis of glycosaminoglycans in bovine cornea. The effect of uridine diphosphate xylose. Biochemical Journal, 120: Klintworth GK & Smith CF (1976). A comparative study of extracellular sulfated glycosaminoglycans synthesized by rabbit corneal fibroblasts in organ and confluent cultures. Laboratory Investigation, 35: Nakayasu K, Gotoh T, Ishikawa T & Kanai A (1996). Glycosaminoglycans in subepithelial opacity after excimer laser keratectomy. Nippon Ganka Gakkai Zasshi, 100:
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